Fig. 1: The FH2 domains of DIAPH1 and 3 directs their actin isoform specificity.

a Schematics of the domain organization of DIAPH1 and 3. The domain boundaries are denoted as the amino acid number in the sequence. DAD diaphanous autoinhibitory domain, DID-DAD interacting domain, FH1 formin homology domain 1, FH2 formin homology domain 2, and CT carboxy-terminal domain that is used in subsequent experiments (amino acids 583–1272 and 540–1171 for DIAPH1 and 3 respectively). b Micrographs of HeLa cells expressing GFP targeted to the surface of mitochondria by a fragment of Tom20 (green), fixed and stained for β- or γ-actin (magenta). Scale bars represent 10 μm. The presented micrographs are representative of three independent experiments. c Relative colocalization, determined by a Manders’ overlap coefficient (tM1) of β- and γ-actin to mitochondria in HeLa cells expressing Tom20-GFP fused to the different CT domains of wildtype DIAPH1 (DIA1), wildtype DIAPH3 (DIA3), DIAPH1 with the DIAPH3 FH2 domain (DIA1 3F) and DIAPH3 with the DIAPH1 FH2 domain (DIA3 1F). Data were presented as mean (solid bar) ± SD (error bars). n = 20 cells analyzed per condition across three independent experiments, *p = 7.25 × 10⁻¹¹, **p = 1.45 × 10⁻¹¹ as calculated by two-sided Mann–Whitney non-parametric tests. d Micrographs of HeLa cells expressing either a Tom20-GFP-DIAPH3-CT fusion protein or a Tom20-GFP-DIAPH3-1F CT (green), fixed and stained for β- or γ-actin (magenta). Scale bars represent 10 μm. The presented micrographs are representative of three independent experiments. e Micrographs of HeLa cells expressing either a Tom20-GFP-DIAPH1-CT fusion protein or a Tom20-GFP-DIAPH1-3F CT (green), fixed and stained for β- or γ-actin (magenta). Scale bars represent 10 μm. White arrowheads point to regions of actin colocalizing to mitochondria. The region boxed is yellow and is magnified in the zoom panel. The presented micrographs are representative of three independent experiments.